Field of the Disclosure
The disclosure relates generally to microscope imaging of tissue and, more particularly, to devices for dual-axes confocal microscopy.
Brief Description of Related Technology
While tremendous advances have been made in technologies for whole body molecular imaging, including PET, SPECT, MRI, and ultrasound, techniques for visualizing biological phenomenon with sub-cellular resolution in optically thick tissue are lacking. In addition, these conventional techniques lack the ability to obtain vertical cross-sectional views of tissue. Take two common forms of cancer, for example, colorectal cancer and breast cancer. Researchers are unable to properly image cancer tissue with vertical cross-sectional views at the sub-cellular level for either of these types of cancer.
Colorectal cancer claims approximately 149,000 new cases annually in the United States, where the rate of incidence is among the highest in the world (48.2 cases/100,000 population in 1998-2004). The average lifetime risk for developing the disease is 1 in 20 in the industrialized world. Yet, despite the widespread availability of colonoscopy, colorectal carcinoma remains the second most common cause of cancer; and the mortality and morbidity associated with this disease is far more common than expected. The miss rate for colonic adenomas, as determined by tandem colonoscopy, has been found to be as high as 22%; and a significant number of cases of colorectal cancers (2.4 cancer/1000 person-years) have been diagnosed within a year following screening colonoscopy. Moreover, there is evidence that up to 25% of spontaneously occurring adenomas arise from sporadically occurring flat or depressed lesions that may be difficult to visualize by conventional white light endoscopy. These lesions frequently contain high grade dysplasia and progress more rapidly through the adenoma-carcinoma sequence than polypoid adenomas. To develop better strategies for risk stratification and early detection, a greater understanding of molecular target expression patterns within the colon tissue over time is needed.
Breast cancer in women is the most common cancer in the U.S. and the second-most common cause of death by cancer. There are approximately 182,000 new cases of invasive breast cancer and about 68,000 new cases of in situ breast cancer each year. Current methods for breast cancer screening include self breast exams, mammography, ultrasound, and MRI. All of these techniques are based on the presence of gross morphological changes in either size or density of malignant lesions for detection. This disease results from multiple genetic and environmental factors, including defects in DNA repair genes, abnormal growth factor synthesis, dysregulated cell signaling, elevated estrogen exposure, and failure of immune surveillance. The presence of molecular changes occur well in advance of structural abnormalities, and more sophisticated imaging methods are needed to improve our understanding of disease mechanisms and to develop more effective screening methods to improve monitoring of treatment efficacy.
One imaging technique for early stage diagnosis is intra-vital imaging of tissue epithelium. Transformed cells that develop into cancer in the colon and breast originate within the epithelium of the mucosa and ducts, respectively, as shown below in FIG. 1. Infra-vital microscopy is a useful tool for studying the molecular mechanisms of epithelial cancer biology in vivo because this technique can be used to directly access this thin, superficial layer of tissue to provide the highest resolution possible with imaging in live animals. A miniature fiber optic instrument can be placed in contact with the tissue surface and collect real time images with sub-cellular resolution. These instruments, for example, can be inserted into the colon and or held by hand onto the breast to perform longitudinal studies in small animal models of cancer. With intra-vital imaging, non-terminal studies can be performed using each animal as its own control. This approach can significantly reduce the number of testing specimens needed and can provide a more robust study design. In addition, this technique can be used to study ligand-receptor interactions and cell tracking behavior in vivo, processes that are difficult to observe with any other imaging modality.
As shown in FIG. 1, normal colonic epithelium transforms to a pre-malignant condition (dysplasia) prior to evolving into carcinoma, viewing the illustration from left to right. Subtle molecular changes develop first in the crypts prior to morphological changes in the tissue. Imaging in the vertical cross-section (plane perpendicular to tissue surface) is the desired orientation for detecting disease because the epithelium differentiates in the vertical direction (basilar to luminal direction). The vertical cross-section provides a global view of the normal and abnormal micro-architectural changes, including the vascular endothelium, in the epithelium with a consistent orientation. This view allows the observer to detect subtle differences associated with the early presence of disease in comparison to that of the horizontal cross-section (parallel to tissue surface).
While useful, intra-vital microscope techniques are limited. One of the challenges in performing high resolution (sub-cellular) imaging in live specimens (e.g., animal models) is the ability to overcome motion artifact, including respiratory displacement, heart beating, and organ peristalsis. Conventional intra-vital microscopes use bulk optic objectives that are fixed to large, stationary platforms. As a result, motion will occur in live animals relative to the objective that appear exaggerated in the relatively small fields-of-view of intra-vital microscopes, typically on the order of several hundred microns. On the other hand, a miniature intra-vital microscope has the size and weight to move relative to the bodily motion of the animal during the imaging session, thus substantially reduce the motion artifact in the images. Fiber coupling allows for the image to be transmitted to the detector. Furthermore, the small size of these instruments provides much greater positioning accuracy of the objective lens onto target organs in the animal.
Recent advances in the development of micro-lenses and scanners have resulted in the development of a number of miniature intra-vital microscopes for high resolution imaging in small animals. These instruments are designed for a size that can be inserted in the colon to evaluate the epithelium of the distal mucosa or hand held against the body wall to image the epithelium of breast ducts. However, these microscopes all use the single axis configuration where the pinhole (fiber) and objective are located along one main optical axis. A high numerical (NA) objective is needed to achieve sub-cellular resolution and maximum light collection, and the same objective is used for both the illumination and collection of light. In order to scale down the dimension of these instruments for small animal imaging, the diameter of the objective must be reduced to ˜5 mm or less. As a consequence, the working distance, imaging depth, and field-of-view are also decreased, as shown by the progression of the 3 different objective diameters (A→B→C) in FIG. 2, with corresponding illustrations of example working distances WDA, WDB, and WDC. The range of sizes is illustrated in the right panel by an objective from the Olympus IV100 where upper element (A) represents the diameter of a conventional objective and the lower reflects that of a miniature objective (C), scale bar 5 mm.
Generally, imaging tissue through the use of light is a very powerful tool, because this modality can achieve sub-cellular resolution in real time, a level of performance that cannot be matched by any other imaging modalities (such as PET scanner, CT scans, MRI, etc.). However, light is highly scattered by tissue, and sophisticated methods are needed to produce clear images. Confocal microscopy is one form of intra-vital microscope that uses a pinhole placed in between the objective lens and the detector to allow only the light that originates from within a tiny volume below the tissue surface to be collected. All other sources of scattered light do not have the correct path to be detected, and thus become “spatially filtered.” This process is known as optical sectioning and can produce a high resolution image from a thin slice of tissue below the surface. These images can be collected at sufficiently fast frame rates to observe biological behavior in small animal models of disease with minimal disturbance from motion artifacts caused by breathing displacements and heart beating. Recent advancements in miniaturization of optics, availability of fiber-optics, and emergence of micro-scanners have allowed for the technique of confocal microscopy to be performed in vivo through medical endoscopes to perform rapid, real-time optical assessment of tissue pathology.
Traditional confocal intra-vital microscopes used a single-axis optical design, while recently some have proposed a dual-axes confocal architecture. For a single-axis design, the pinhole (i.e., the single mode optical fiber) and objective are located along the same optical axis. As a result, a high numerical aperture (NA) objective is needed to achieve sub-cellular resolution, limiting the working distance as discussed above in reference to FIG. 2. As a consequence, the single-axis configuration uses a scanning mechanism (mirror) that is placed on the pinhole side of the objective, or in the pre-objective position, and this design cannot be scaled down in dimension without loss of working distance or field-of-view. Furthermore, much of the light that is scattered by the tissue present between the objective and focal volume (dashed lines) is collected with the high NA objective, reducing the dynamic range of detection. See, e.g., FIG. 3A.
The more recent dual-axes architecture, shown in FIG. 3B, uses two fibers and low NA objectives for separate illumination and collection of light, using the region of overlap between the two beams (focal volume) to achieve sub-cellular resolution. The low NA objectives create a long working distance so that the scan mirror can be placed on the tissue side of the lens, or in the post-objective position. Very little of the light that is scattered by tissue along the illumination path (dashed lines) is collected by the low NA objective collection objective, thus the dynamic range is significantly improved. Consequently, theoretically optical sections can be collected in both vertical (V) and horizontal (H) planes with dual axes, as compared to horizontal only for single axis. Vertical cross-sections show the relationship among tissue microstructures as they vary with depth, and are the preferred view of pathologists. The single-axis configuration does not have sufficient dynamic range to provide this view.
In intra-vital microscopy, scanning of the focal volume is performed to create an image. In the single-axis architecture, the limited working distance requires that the scan mirror be placed in the pre-objective position, as shown in FIG. 4A, i.e., before the incident light from the fiber/pinhole hits the objective. The mirror is used for scanning which steers the beam at various angles to the optical axis (dashed line) and introduces off-axis aberrations that distort the focal volume. In addition, because the FOV is proportional to the scan angle and the focal length, the diameter of the objective limits the maximum scan angle. As this dimension is reduced, the focal length and FOV are also diminished (arc-line). In the dual-axes configuration, the low NA objectives create a long working distance that allows for the scanner to be placed in the post-objective position. This design feature is useful for scaling the size of the instrument down to millimeter dimensions for in vivo imaging in small animal models without losing performance. As shown in FIG. 4B, the illumination light is always incident on-axis to the objective. In this scanning geometry, the scan mirror can sweep a diffraction-limited focal volume over an arbitrarily large FOV (arc-line), limited only by the maximum deflection angle of the mirror.
Recently, a dual-axes confocal architecture was implemented in a 5 mm diameter instrument package, as shown in FIG. 5A. The scanhead uses a replicated parabolic mirror as the low NA focusing objective, and a MEMS (micro-electro-mechanical-systems) mirror to perform scanning in the horizontal (XY) plane. Axial (Z-axis) translation is performed with a stepper (micro-) motor. Illumination and collection of light is delivered separately by two single mode optical fibers, and control wires contained within an umbilical provide power and scanning signals to the MEMS mirror and micro-motor. This instrument can be either inserted into the colon or held by hand against the breast of genetically engineered mice, as shown by FIG. 5B. The instrument can collect fluorescence images in horizontal cross-sections from a depth of z=0 to 500 μm in 3 μm intervals. The simple Z-axis scanner used in this design is limited and requires large amounts of time (on the order of 50 seconds) to perform a full Z-axis scan. Such limited scannability in the Z-axis means that the conventional dual-axes configuration is not sufficiently fast to collect vertical cross-sectional images.
Thus, there is a need for a dual-axes scanning assembly that offers sufficient Z-axis and XY-plane scanning, in a configuration that can achieve high scan rates (such as real time scan rates) over a volume of tissue. Furthermore, it is desirable to use such a device for more accurate pathology recognition and earlier detection.